The invention relates to compounds, methods and kits for the determination of hydrophobic drugs such as, for example, immunosuppressant drugs, in samples, such as patient samples, known or suspected to contain one or more of such hydrophobic drugs.
The body relies upon a complex immune response system to distinguish self from non-self. At times, the body's immune system must be controlled in order to either augment a deficient response or suppress an excessive response. For example, when organs such as kidney, heart, heart-lung, bone marrow and liver are transplanted in humans, the body will often reject the transplanted tissue by a process referred to as allograft rejection.
In treating allograft rejection, the immune system is frequently suppressed in a controlled manner with drug therapy. Immunosuppressant drugs are carefully administered to transplant recipients in order to help prevent allograft rejection of non-self tissue. Two most commonly administered immunosuppressive drugs to prevent organ rejection in transplant patients are Cyclosporine (CSA) and FK-506 (FK or tacrolimus). Another drug that finds use as an immunosuppressant in the United States and other countries is sirolimus, also known as rapamycin. Derivatives of sirolimus are also said to be useful as immunosuppressants. Such derivatives include, for example, Everolimus, and the like.
The side effects associated with some immunosuppressant drugs can be controlled in part by carefully controlling the level of the drug present in a patient. Therapeutic monitoring of concentrations of immunosuppressant drugs and related drugs in blood is required to optimize dosing regimes to ensure maximal immunosuppression with minimal toxicity. Although immunosuppressant drugs are highly effective immunosuppressive agents, their use must be carefully managed because the effective dose range is often narrow and excessive dosage can result in serious side effects. On the other hand, too little dosage of an immunosuppressant can lead to tissue rejection. Because the distribution and metabolism of an immunosuppressant drug can vary greatly between patients and because of the wide range and severity of adverse reactions, accurate monitoring of the drug level is essential.
Most whole blood assays for immunosuppressant drugs require a manual step to extract the drug from blood constituents using organic solvents. As a result, the drug molecules are extracted into a relatively clean solution in which plasma proteins and lipoprotein particles as well as most other molecules are removed. Therefore, the binding of assay antibody to the drug occurs in the absence of most endogenous substances in these assays. However, in a homogenous assay where there is no manual extraction or separation of the drug from blood constituents, an antibody for the immunosuppressant drug has to detect the drug in the presence of most or all blood constituents, which might interfere the binding of the antibody to the immunosuppressant drug and/or its analog.
The present inventors have recognized that hydrophobic drugs, such as immunosuppressant drugs, are absorbed by cholesterol-rich lipoprotein particles (such as low density lipoprotein (LDL) and high density lipoprotein (HDL), etc. The drug is absorbed in such a way that a portion or all of the drug becomes inaccessible to the detection antibody in an assay for the drug, resulting in a decrease in the amount of detectable drug in the assay. Because hyperlipidemia is common in transplant patients, an assay for the determination of an immunosuppressant drug needs to be robust to lipoprotein interference.
There is, therefore, a continuing need to develop fast and accurate diagnostic methods to measure levels of immunosuppressant drugs or derivatives thereof in patients. The methods should be fully automated and be accurate even when conducted on whole blood samples with no-extraction using a homogenous assay where an antibody employed in the assay has to detect the drug in the presence of most, if not all, blood constituents.